Cell culture and tissue engineering systems with controlled environmental zones

ABSTRACT

An automated cell culture and tissue engineering system comprising defined and separate environmental zones provide for increased control and maintenance of the internal environment of the system such that the temperature, air flow and gases surrounding the bioreactor module form one zone that is maintained separately to a second zone formed surrounding the reagent fluid reservoir. The system further comprises means for elimination and/or management of condensation within the second zone of the system.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of U.S. application Ser. No.16/727,367, filed Dec. 26, 2019, which claims benefit of U.S.Provisional Application No. 62/785,998, filed Dec. 28, 2018, thedisclosures of each of which are incorporated by reference herein intheir entireties.

FIELD

The invention relates to tissue engineering systems and methods forautomated cell culture and tissue engineering that include uniformoperational environmental zones to provide more consistent biologicalprocesses. Such systems and methods find use in a variety of clinicaland laboratory settings.

BACKGROUND

Cell culture automation is a desired trend for providing scalability formass production, decreasing variability in culture, decreasing risks ofculture contamination as well as many economical cost and time framebenefits related to generation of cell or tissue based implants forclinical therapies and cell based assay systems for diagnosticevaluations.

Automated cell culture protocols used for more complex biologicalprocesses, such as for example autologous patient treatment, however maybe more complex requiring more precise operational control. Forautologous patient treatment, successful biological culture is criticaland thus each operational aspect must be strictly adherent to a specificprotocol. For example, while initial culture media can be programmed fordelivery to cell cultures at suitable temperatures such as 37° C.,maintaining a strict adherence to this temperature throughout an entirecell culture process that may be days long, proves more difficult. Manycurrently used automated systems providing cell incubation capabilityexperience thermal challenges such as: trying to achieve and maintainoverall thermal uniformity; changes in air temperatures due to externaloperator access; maintaining refrigerated reagent storage; and thedevelopment of condensation within the housing which may lead topotential microbial contamination.

Refrigerated storage of reagents is desired to avoid theirdeterioration, however, refrigerated reagent storage can negativelyaffect the thermal performance and stability of the cell bioreactormaking it more difficult to maintain the desired elevated temperaturerequired for cell culture. Furthermore, the reduction of the temperatureof the storage environment for process reagents to refrigeratedtemperatures inevitably generates condensation. Condensation forms whenthe air is chilled below the dew point causing the water vapor in theair to condense into a liquid form, especially on surfaces that are coldrelative to the surroundings. Warm, humid air may come into contact withthe colder surface of the storage environment when the system is openedto load the reagents. This condensation can be problematic if the volumeof condensate results in handling issues or contamination issues withinthe storage environment.

Gas concentrations can also significantly influence biologicalperformance as gases such as CO₂ are used to modulate the pH of activecultures. In the event that the culture system experiences restrictedaccess to the buffering role of delivered CO₂, there is the prospect ofinhomogeneous pH control across the culture zone. Thus gas concentrationuniformity is also required in cell culture and tissue engineeringsystems.

Accordingly there is still a continual need to improve aspects ofautomated cell culture and tissue engineering systems such as providinghigher fidelity of environmental control within the system.

The discussion of the background herein is included to explain thecontext of the inventions described herein. This is not to be taken asan admission that any of the material referred to was published, known,or part of the common general knowledge as of the priority date of anyof the claims.

SUMMARY

Herein described are cell culture and tissue engineering systems andmethods that include uniform operational environments to provide moreconsistent control over biological processes. Uniform operationalenvironments relating to airflow, temperature, gas control andcondensation control are integrated within the systems and methodsdescribed herein.

Automated cell culture and tissue engineering systems as describedherein are configured to generate, adjust and maintain controlledisolated environmental zones for proper operation of a cell culturecassette and thus the biological process that is underway. Temperatures,humidity levels and gas concentrations are controlled. Temperaturefluctuations and humidity variations are minimized.

Surprisingly, two distinct temperature zones are created and maintainedin the cassette receiving area of the system where the cell culturecassette is installed and operated. The bioreactor module component ofthe cassette is retained in a warm zone while the reagents fluidreservoir component is retained in a cold zone during operation of thesystem. The warm zone comprises a re-circulating warm high airflow pathsurrounding the bioreactor module that is generated by an adjacentseparate heating assembly. The warm circulating air also permeates theculture aspect of the cassette through ventilation slots. This issimilar in concept to the slots in the reservoir. The cold zonecomprises a circulating cold ducted airflow path that both surrounds andpartially penetrates (flows through) a portion of the reagents fluidreservoir. The cold airflow path is generated by an adjacent coldthermal assembly. The cold zone also contains a condensation controlmeans for the control and removal of condensation within the cold zone.

Both the warm zone and cold zone further comprise a segregatedindependently controlled gas environment. In the warm zone this helps toprovide for specific gas concentrations that influence dissolved gasespresent in cell culture media such as oxygen and carbon dioxide throughfluid surface gas exchange. The adjustment and/or maintenance ofdissolved gases is influential in terms of biological performance,including aspects such as the delivery of oxygen and the maintenance ofa target pH for the cell culture. The control of dissolved gases withinthe culture media is achieved through the recirculation of culture mediaacross a gas exchange membrane (such as silicone tubing) whereby theconcentration of dissolved gases within the culture fluid responds tothe concentration gradient between the fluid and the surrounding gaseousenvironment. By adjusting the environment, the level of dissolved gaseswithin the culture fluid is simultaneously adjusted.

The automated cell culture and tissue engineering system is configuredwith a movable thermal barrier assembly that, during installation of acell culture cassette, serves to lock the cassette within the systemoperational interface and in doing so, forms the warm zone and the coldzone and keeps these two zones thermally and physically separated.Insulating mechanisms are provided to ensure that the warm zone and coldzone are insulated from each other and do not influence either the cellculture or the properties of the stored reagents. The movable thermalbarrier assembly creates and defines portions of the borders of each ofthe warm zone and the cold zone.

The system heating and cooling assemblies, as well as operationalrobotics are contained/positioned separate to that of the cassettereceiving area of the system, which is beneficial not to interfere withwarm airflow or cold airflow paths and their function. Further, theheating assembly is segregated to be separate and insulated from thecooling assembly. The system heating assembly is configured to generateand regulate the temperature of the continuous warm high airflow pathfor several days as is required and further can quickly adjust for anytemperature drop that may occur due to the entrance of cooler roomtemperature air during opening and inspection of the system. Theconfiguration and shape of the system helps to provide for a warmairflow path to be directed solely at the bioreactor module andcontinually circulate around and through it. The cold thermal assemblyis configured to continually remove heat as airflow in the cold zone iscontinually drawn through a cold thermal assembly and the airtemperature is reduced. The cold zone is configured to have a channeledairflow, that is, the cold airflow path follows structural featuresincluding airflow channels, airflow baffles and air flow vents to helpcarry the cooled air through the cold zone around and partially throughthe reagents fluid reservoir and further through an optional adjacentcold reservoir external to the reagents fluid reservoir. Thesestructural features help to prevent and minimize any blockage of thereturn air (by filled fluids bags) traveling toward to the cold thermalassembly.

The provision of a separate warm zone and separate cold zone minimizesthe amount and location of condensation that may form within the systemas the system is only subjected to condensation in the cold zone whichhas a mechanism to effectively prevent, control and remove condensation.

The invention in aspects comprises an automated cell culture and tissueengineering system that comprises a closed automated cell culturecassette for the one of more of cell source isolation, cellproliferation/expansion, cell differentiation, cell isolation, celllabelling, cell purification, cell washing and cell seeding ontoscaffolds for tissue formation (product formation). In aspects, thecells are mammalian. In further aspects, human cells. The type of cellor tissue is not limiting. In one non-limiting example, pluripotent stemcells, such as embryonic stem cells and induced pluripotent stem (iPS)cells, may be cultured and expanded for cell replacement therapy.

The automated cell culture cassette is in aspects a closed, single-usedisposable cassette comprising one or more sterile bioreactor modulesfluidly connected to a reagent fluids reservoir. The sterile bioreactoris loaded with desired cells and/or tissue and connected to the reagentfluid reservoir which is preloaded to contain the required fluidreagents. The sterility of the cassette is maintained throughout.

The automated cell culture cassette is operatively employed within anautomated cell culture and tissue engineering system along with adedicated software program to deliver and track a desired process(es).Suitable non limiting automated cell culture and tissue engineeringsystems are described in U.S. Pat. Nos. 8,492,140; 9,701,932; 9,534,195;9,499,780; and 9,783,768 (the contents of each of these U.S. patents isincorporated by reference in their entireties).

Embedded sensors within the cell culture cassette, provide real-timebiofeedback and enable automatic adjustment in bioprocessing toaccommodate natural variations in cell source behaviour. The entirebioprocess is contained within the disposable cassette to ensure maximumpatient and operator safety and to streamline logistics. Further, inorder to successfully support multiple biological steps in a cellprocess sequence, the cassette bioreactor(s) are integrated incombination with biosensor feedback within one or more interlinkedbioreactors, to provide a highly intuitive system with precise controlat each cell and tissue stage. This comprehensive level of automationenable technically feasible and economical scale-up, patient-scale cellmanufacturing capabilities and allows streamlined production of celltherapies under good manufacturing practice (GMP) conditions thusmeeting the unique challenges of different autologous and allogeneicclinical applications of cell and tissue therapy.

Advantageously, the cell culture cassette is installed and operationallyretained within the cell culture and tissue engineering system housingin a manner that the bioreactor module resides solely in the distinctwarm zone and the reagents fluid reservoir resides solely in a distinctcold zone. The installation of the cell culture cassette into the systemis via the actuation and locking of a movable thermal barrier assemblythat environmentally isolates the first thermal zone from the secondthermal zone with respect to temperature, gases and humidity. The cellculture cassette comprises the bioreactor module with attached reagentsfluids reservoir. While this combination into a single cell culturecassette is more user friendly, it poses more of a challenge withrespect to creating separate and distinct environmental zones for thebioreactor module and for the reagents fluid reservoir versus a moresimplistic design based on physically separate bioreactor module andreservoir that can be located in separate environmental zones.

The invention provides dedicated airway paths within the warm zone andthe cold zone ensuring controlled distribution of temperature/gas isprovided about the bioreactor module housing the cell culture(s) andabout the reagents fluid reservoir in a manner to preclude points ofdistortions of uniformity in each zone.

In aspects of the invention is a cell culture and tissue engineeringsystem comprising two distinct independent thermal airflows, a firstairflow comprising a high velocity warm airflow for directing at andaround a bioreactor module of a cell culture cassette, and a secondairflow comprising a cold airflow for circulating around and through areagents fluid reservoir operatively connected to the bioreactor module,wherein said first airflow and said second airflow are separatesubstantially (these zones in embodiments are not hermetically sealedrelative to each other) and cannot intermingle.

In aspects the first airflow and said second airflow are containedwithin a cell culture cassette receiving area of said system.

In aspects, the cell culture cassette receiving area is separate fromoperational, heating and cooling assemblies of the system.

In aspects the cell culture cassette receiving area defines a warm zonethat comprises the high velocity warm airflow.

In aspects the warm zone comprises substantially homogeneous temperaturewithin the warm zone.

In aspects the cell culture cassette receiving area defines a cold zonethat comprises the cold airflow, said cold zone positioned beneath thewarm zone.

In aspects the cold zone comprises a means for reducing or eliminatingcondensation.

In aspects a thermal platform separates the warm zone from the coldzone. In aspects, the thermal platform contains seals.

In aspects of the invention is a method for maintaining a controlledthermal environment for biological processes within a bioreactor moduleof a cell culture cassette, the method comprising:

directing a first airflow comprising a high velocity warm airflow at andaround the bioreactor module, and simultaneously circulating a cold airflow around and through a reagents fluid reservoir operatively connectedto the bioreactor module,

wherein said first airflow and said second airflow are separate andcannot intermingle.

According to an aspect of the invention is a cell culture and tissueengineering system comprising a thermal zone architecture for providinga more consistent and controlled environment for facilitating biologicalprocesses, wherein the system comprises:

a distinct warm zone compartment that retains a bioreactor module whilecontinuously circulating a high velocity warm airflow path around anddirected at and around the bioreactor module; and

a distinct cold zone compartment that retains a reagents fluid reservoirfunctionally connected to the bioreactor module, while continuouslycirculating a cold airflow path around and through the reagents fluidreservoir.

In aspects, the distinct cold zone compartment further comprises a meansfor reducing or eliminating condensation.

In aspects, the warm zone compartment further comprises a distinct gasenvironment to that of the cold zone compartment.

According to a further aspect of the invention is an automated systemfor cell culture and tissue engineering that retains a cell culturecassette in two distinct temperature and gas controlled environments,wherein a biological reactor component of the cassette is operativelyretained in a substantially homogeneous warm airflow zone withcontrolled gas concentrations, and wherein a reagents fluid reservoircomponent of the cassette resides in a substantially homogeneous coldairflow zone, wherein the warm airflow zone is distinctly separated fromthe cold airflow zone, and wherein the cold airflow zone furthercomprises a means for preventing or eliminating undesirable moistureaccumulation therein.

According to a further aspect of the invention is a cell culture andtissue engineering system for receiving and operationally supporting anautomated cell culture cassette in a more consistently controlledenvironment for biological processes, the cell culture cassettecomprising a bioreactor module and a reagents fluid reservoir, thesystem comprising:

a warm zone configured for circulating a warm airflow path surroundingthe bioreactor module;

a cold zone configured for circulating a cold airflow path surroundingthe reagents fluid reservoir; and

a movable thermal barrier assembly for thermally isolating said warmzone from said cold zone upon installation of the cell culture cassette,and for securing the bioreactor module solely within the warm zone andthe reagents fluid reservoir solely within the cold zone.

According to a further aspect of the invention is a cell culturecassette comprising:

-   -   a bioreactor module having a bottom part attached with a        reagents fluid reservoir;    -   the reagents fluid reservoir comprising a fluids bag container        having open air ducts located on front and back walls of the        reservoir.

In aspects, the fluids bag container comprises a roof and floor, theroof comprising baffles extending downwardly.

In aspects, the cassette further comprises a layer of thermal insulationpositioned in between the bottom of the bioreactor module and the roofof the fluids bag container, said layer of thermal insulation insulatingagainst migration of heat from the bioreactor module.

In aspects, the reagents fluid reservoir is attached via portconnections positioned on the roof of said fluids bag container andunobstructed by said layer of thermal insulation.

In aspects, the reagents fluid reservoir further comprises snap tabs forattaching to the bioreactor module.

According to a further aspect of the invention is a reagents fluidreservoir for connection to a bioreactor module, the reagents fluidreservoir comprising a fluids bag container having open air ductslocated on front and back walls of the reservoir.

In aspects, the fluids bag container comprises a roof and floor, theroof comprising baffles extending downwardly.

According to an aspect of the invention is an automated cell culture andtissue engineering system for receiving and operationally supporting anautomated cell culture cassette in a more consistently controlledenvironment for biological processes, the cell culture cassettecomprising a bioreactor module and a reagents fluid reservoir, thesystem comprising:

a warm zone configured for circulating a tangential warm airflow pathsurrounding the bioreactor module;

a cold zone configured for circulating a tangential cold airflow pathsurrounding the reagents fluid reservoir; and

a movable thermal barrier assembly for thermally isolating said warmzone from said cold zone upon installation of the cell culture cassette,and for securing the bioreactor module solely within the warm zone andthe reagents fluid reservoir solely within the cold zone.

In any of the aforementioned aspects, the system and method may compriseone or more controllers and associated software, sensors, and userinterface.

Non-limiting aspects are as follows:

1A. A cell culture and tissue engineering system comprising two distinctindependent thermal airflows, a first airflow comprising a high velocitywarm airflow for directing at and around a bioreactor module of a cellculture cassette, and a second airflow comprising a cold airflow forcirculating around and through a reagents fluid reservoir operativelyconnected to the bioreactor module, wherein said first airflow and saidsecond airflow are separate and cannot intermingle.1B. The system of claim 1A, wherein said first airflow and said secondairflow are contained within a cell culture cassette receiving area ofsaid system.1C. The system of claim 1B, wherein said cell culture cassette receivingarea defines a warm zone that comprises the high velocity warm airflow.1D. The system of claim 1C, wherein said warm zone comprisessubstantially homogeneous temperature within the warm zone.1E. The system of claim 1B, 1C or 1D, wherein said cell culture cassettereceiving area defines a cold zone that comprises the cold airflow, saidcold zone positioned beneath the warm zone.1F. The system of claim 1E, wherein said cold zone comprises a means forreducing or eliminating condensation.1G. The system of claim 1F, wherein a thermal platform separates thewarm zone from the cold zone.1H. A method for maintaining a controlled thermal environment forbiological processes within a bioreactor module of a cell culturecassette, the method comprising:

directing a first airflow comprising a high velocity warm airflow at andaround the bioreactor module, and simultaneously circulating a cold airflow around and through a reagents fluid reservoir operatively connectedto the bioreactor module,

wherein said first airflow and said second airflow are separate andcannot intermingle.

1J The method of claim 1H using the system of any one of claims 1A to1H.2A. A cell culture and tissue engineering system comprising a thermalzone architecture for providing a more consistent and controlledenvironment for facilitating biological processes, wherein the systemcomprises:

a distinct warm zone compartment that retains an bioreactor module whilecontinuously circulating a high velocity warm airflow path around anddirected at and around the bioreactor module; and

a distinct cold zone compartment that retains a reagents fluid reservoirfunctionally connected to the bioreactor module, while continuouslycirculating a cold airflow path around and through the reagents fluidreservoir.

2B. The system of claim 2A, wherein the distinct cold zone compartmentfurther comprises a means for reducing or eliminating condensation.2C. The system of claim 2A or 2B, wherein said warm zone compartmentfurther comprises a distinct gas environment to that of the cold zonecompartment.2D A method for maintaining a controlled environment for biologicalprocesses within a bioreactor and for maintaining fluids required forthe bioreactor at a cool temperature for stability, the methodcomprising the use of the system of any one of claims 2A to 2C.3A. An automated system for cell culture and tissue engineering thatretains a cell culture cassette in two distinct temperature and gascontrolled environments, wherein a biological reactor component of thecassette is operatively retained in a substantially homogeneous warmairflow zone with controlled gas concentrations, and wherein a reagentsfluid reservoir component of the cassette resides in a substantiallyhomogeneous cold airflow zone, wherein the warm airflow zone isdistinctly separated from the cold airflow zone, and wherein the coldairflow zone further comprises a means for preventing or eliminatingundesirable moisture accumulation therein.3B. A method for maintaining a controlled environment for biologicalprocesses within a bioreactor and for maintaining fluids required forthe bioreactor at a cool temperature for stability, the methodcomprising the use of the system of claim 3A.1. A cell culture and tissue engineering system for receiving andoperationally supporting an automated cell culture cassette in a moreconsistently controlled environment for biological processes, the cellculture cassette comprising a bioreactor module and a reagents fluidreservoir, the system comprising:

a warm zone configured for circulating a warm airflow path surroundingthe bioreactor module;

a cold zone configured for circulating a cold airflow path surroundingthe reagents fluid reservoir; and

a movable thermal barrier assembly for thermally isolating said warmzone from said cold zone upon installation of the cell culture cassette,and for securing the bioreactor module solely within the warm zone andthe reagents fluid reservoir solely within the cold zone.

2. The cell culture and tissue engineering system of claim 1, whereinsaid cold zone further comprises a condensation control means forminimizing and eliminating undesirable moisture accumulation therein.3. The cell culture and/or tissue engineering system of claim 1 or 2,wherein said warm zone and said cold zone each comprise a substantiallysegregated gas environment.4. The cell culture and tissue engineering system of claim 1, 2 or 3,said system having a housing comprising an outer shell cover and aninner shell body, wherein the outer shell cover encloses a front openingof the inner shell body in an enveloped manner when the system isclosed.5. The cell culture and tissue engineering system of claim 5, whereinsaid outer shell cover is connected to the inner shell body for rotationwhile the inner shell body remains stationary, the outer shell coverrotates along an outer arc of the inner shell body to expose the frontopening of the inner shell body for access thereto and stops rotatingwhen the outer shell cover nests the inner shell body.6. The cell culture and tissue engineering system of claim 4 or 5,wherein said front opening of the inner shell body comprises a peripherywith an inflatable sealing means for a sealing engagement with an insidesurface of the outer shell body when the system is closed.7. The cell culture and tissue engineering system of claim 6, whereinsaid periphery comprises a U shaped channel for retaining saidinflatable sealing means.8. The cell culture and tissue engineering system of claim 7, whereinthe sealing means comprises an elastomeric tube that fits within the Ushaped channel, the elastomeric tube being substantially flat when thesystem is open and the sealing means activated upon closing the systemto introduce an inflation pressure into the cavity of the inflatableseal causing displacement of the seal to effect a positive seal betweenthe outer shell cover and the front opening of the inner shell body.10. The cell culture and tissue engineering system of claim 9, whereinsaid outer shell cover is configured as an arc-shaped body that helps todirect the circulating warm airflow path surrounding the bioreactormodule and also helps to direct the circulating cold airflow pathsurrounding the reagents fluid reservoir.11. The cell culture and tissue engineering system of claim 10, whereinsaid arc-shaped body comprises a plurality of thermal cells to act as anexterior thermal barrier.12. The cell culture and tissue engineering system of any one of claims4 to 11, wherein the movable thermal barrier assembly is disposed on anoperational robotics interface positioned within the front opening ofthe inner shell body.13. The cell culture and tissue engineering system of claim 12, whereinsaid operational robotics interface is connected to associated internalrobotics and comprises valve actuators, peristaltic pumps and relatedcontrol systems for mating with corresponding connections on the cellculture cassette.14. The cell culture and tissue engineering system of any one of claims4 to 11, wherein said movable thermal barrier assembly comprises:

-   -   a pair of spaced apart levered arms internally connected at        either side of the operational robotics interface that support a        central thermal platform with an associated upper handrail        15. The cell culture and tissue engineering system of claim 14,        wherein said movable thermal barrier assembly is movable from a        first raised position for installing the cell culture cassette,        to a second lowered position that locks and retains the cell        culture cassette against the operational robotics interface for        accurate alignment and retention thereto and simultaneously        isolating the warm zone from the cold zone.        16. The cell culture and tissue engineering system of claim 15,        wherein said upper handrail is sized to closely conform to        dimensions of the cell culture cassette and provide a gripping        means to lock the cassette into position for operation or to        raise the thermal platform.        17. The cell culture and tissue engineering system of claim 14,        15 or 16, wherein recesses are provided on the operational        robotics interface adjacent said levered arms for providing        operator controlled access to open the assembly.        18. The cell culture and tissue engineering system of any one of        claims 14 to 17, wherein said thermal platform extends forward        and radiates from around the cell culture cassette to the outer        shell cover.        19. The cell culture and tissue engineering system of claim 18,        wherein the thermal platform comprises a flexible outer seal at        its front outer boundary to accommodate relative motion between        the thermal platform and the outer shell cover and a flexible        inner seal that is directly adjacent to the cell culture        cassette.        20. The cell culture and tissue engineering system of claim 19,        wherein the flexible inner seal accommodates mounting tolerances        of the cell culture cassette.        21. The cell culture and tissue engineering system of claim 19        or 20, wherein said flexible outer seal and the said flexible        inner seal are elastomeric and impermeable to vapor.        22. The cell culture and tissue engineering system of claim 21,        wherein said flexible outer seal and the said flexible inner        sear are wiper-style seals.        23. The cell culture and tissue engineering system of any one of        claims 14 to 22, wherein when in the locked position the thermal        platform comprises part of the lower surface boundary of the        warm zone and part of the upper surface boundary of the cold        zone.        24. The cell culture and tissue engineering system of any one of        claims 14 to 18, wherein the inner shell body comprises a floor        supporting a tray with forward cantilevered ledge that supports        part of a bottom surface of the cell culture cassette.        24a. The cell culture and tissue engineering system of claim 24,        wherein an airflow duct is defined by the floor and the tray.        25. The cell culture and tissue engineering system of any one of        claims 4 to 24, wherein a heating assembly is provided in an        upper section of the inner shell body generates warm air        directed at the bioreactor module.        26. The cell culture and tissue engineering system of claim 25,        wherein the heating arrangement comprises a high capacity linear        fan operably connected to a heated airflow director to generate        and direct a high velocity warm airflow path.        27. The cell culture and tissue engineering system of claim 26,        wherein the high tangential warm airflow path surrounding the        bioreactor module promotes uniformity of temperature of the warm        zone and thus of biological processes within the bioreactor.        28. The cell culture and tissue engineering system of claim 27,        wherein the high tangential velocity warm airflow path        surrounding the bioreactor module helps to maintain the        bioreactor internal temperature at about 35° C., at about 36°        C., at about 37° C., at about 38° C. or at about 39° C.        28a. The cell culture and tissue engineering system of claim 28,        wherein gases are selectively introduced into the warm zone.        28b. The cell culture and tissue engineering system of claim        28a, wherein gases comprise one or more of oxygen, carbon        dioxide and nitrogen.        28c. The cell culture and tissue engineering system of claim 28a        or 28b, wherein the warm zone comprises sensors in the in the        warm airflow path for monitoring the gases.        28d. The cell culture and tissue engineering system of claim        28c, wherein the sensors are operatively connected to a PID        (proportional integral derivative) controller providing feedback        control of gases.        29. The cell culture and tissue engineering system of any one of        claims 4 to 28, wherein a cold thermal assembly forms the rear        of the inner shell body and generates and controls the cold zone        airflow path surrounding the reagent fluid reservoir for        enhanced reagent stability.        30. The cell culture and tissue engineering system of claim 29,        wherein said cold thermal assembly comprises a cold sink array        that comprises a plurality of cold sinks compressed with a        peltier device to a hot sink for transfer of heat to the hot        sink.        31. The cell culture and tissue engineering system of claim 30,        wherein compression is spring compression utilizing a Peltier        solid-state device comprising an array of spring compression        bolts each comprising coil springs, the hot sink acting as a        heat conductive path for heat removal.        32. The cell culture and tissue engineering system of claim 31,        wherein said Peltier solid-state device functions to pump heat        from the cold sinks to the hot sink for subsequent heat transfer        to an ambient environment.        33. The cell culture and tissue engineering system of any one of        claims 30 to 32, wherein each cold sink array is segmented into        functional units comprising a cold sink and associated axial        fan.        34. The cell culture and tissue engineering system of claim 33,        wherein the axial fan has an axial flow configuration.        35. The cell culture and tissue engineering system of claim 33        or 34, wherein the cold sink comprises a vertical fin structure.        36. The cell culture and tissue engineering system of any one of        claims 29 to 35, further comprising thermal insulation to        inhibit heat from returning to the cold sink from the hot sink.        37. The cell culture and tissue engineering system of any one of        claims 2 to 36, wherein said condensation control means        comprises a moisture transport material enclosed within a duct        connected to the hot sink, the moisture transport material        collecting and moving any condensate that may form and that        travels down the cold sink and through to the hot sink where it        is evaporated.        38. The cell culture and tissue engineering system of claim 37,        wherein said condensation control means minimizes and eliminates        undesirable microbial contamination from moisture accumulation.        39. The cell culture and tissue engineering system of any one of        claims 29 to 38, wherein the cold thermal assembly can be        unhinged from the bottom of the inner shell body to hang in an        open configuration for cleaning.        40. The cell culture and tissue engineering system of claim 39,        wherein the cold sink array and associated axial fan may be        uncoupled for cleaning.        41. The cell culture and tissue engineering system of claim 40,        wherein the hot sink fan and associated cowling may be unlatched        from an upper portion of the inner shell body and raised into an        open configuration for cleaning.        42. The cell culture and tissue engineering system of any one of        claims 1 to 41, wherein the reagent fluids reservoir is fluidly        connected to the bioreactor module via top mounted port        connections for mating with ports on an underside of the        bioreactor module.        43. The cell culture and tissue engineering system of claim 42,        wherein the reagent fluids reservoir further comprises snap tabs        for reversible attachment to the bioreactor module.        44. The cell culture and tissue engineering system of claim 43,        wherein the reagent fluids reservoir accommodates reagents        required for biological processes and accommodates the retention        of waste products eliminated from the bioreactor module.        45. The cell culture and tissue engineering system of claim 44,        wherein the reagent fluids reservoir comprises a fluid bags        container for storing a plurality of separate reagents and for        storing the waste products.        46. The cell culture and tissue engineering system of claim 45,        wherein said plurality of separate reagents are stored in        reagent bags.        47. The cell culture and tissue engineering system of claim 46,        wherein the waste products are stored in reagent bags.        48. The cell culture and tissue engineering system of claim 46        or 47, wherein said reagent bags are flexible reagent bags.        49. The cell culture and tissue engineering system of any one of        claims 44 to 48, wherein the reagent fluids reservoir further        comprises open air ducts located at the top of front and back        walls such that the open air ducts are positioned above said        reagent bags providing cold airflow directly above the reagent        bags.        50. The cell culture and tissue engineering system of claim 49,        further wherein the reagent fluids reservoir comprises a roof        with downwardly extending baffles to help direct the cold        airflow above and across the reagent bags.        51. The cell culture and tissue engineering system of claim 50,        wherein said cell culture cassette further comprises a layer of        thermal insulation positioned in between said bioreactor module        and said reagent fluids reservoir, said layer of thermal        insulation insulating against any migration of heat from the        bioreactor module.        52. A cell culture cassette comprising:    -   a bioreactor module having a bottom part attached with a        reagents fluid reservoir;    -   the reagents fluid reservoir comprising a fluids bag container        having open air ducts located on front and back walls of the        reservoir.        53. The cell culture cassette of claim 52, wherein the fluids        bag container comprises a roof and floor, the roof comprising        baffles extending downwardly.        54. The cell culture cassette of claim 52 or 53, wherein the        cassette further comprises a layer of thermal insulation        positioned in between the bottom of the bioreactor module and        the roof of the fluids bag container, said layer of thermal        insulation insulating against migration of heat from the        bioreactor module.        55. The cell culture cassette of any one of claim 53 or 54,        wherein the reagents fluid reservoir is attached via port        connections positioned on the roof of said fluids bag container        and unobstructed by said layer of thermal insulation.        56. The cell culture cassette of claim 55, wherein the reagents        fluid reservoir further comprises snap tabs for attaching to the        bioreactor module.        57. An automated cell and tissue culture engineering system        comprising the cell culture cassette of any one of claims 52 to        56.        57a. The automated system of claim 57, wherein the cassette        comprises one or more sensors linked to a logic means.        58. A reagents fluid reservoir for connection to a bioreactor        module, the reagents fluid reservoir comprising a fluids bag        container having open air ducts located on front and back walls        of the reservoir.        59. The reagents fluid reservoir of claim 57, further wherein        the fluids bag container comprises a roof and floor, the roof        comprising baffles extending downwardly.        60. A cell culture cassette comprising a bioreactor module and a        reagents fluid reservoir according to claim 58 or 59.        61. An automated cell culture and tissue engineering system for        receiving and operationally supporting an automated cell culture        cassette in a more consistently controlled environment for        biological processes, the cell culture cassette comprising a        bioreactor module and a reagents fluid reservoir, the system        comprising:

a warm zone configured for circulating a tangential warm airflow pathsurrounding the bioreactor module;

a cold zone configured for circulating a tangential cold airflow pathsurrounding the reagents fluid reservoir; and

a movable thermal barrier assembly for thermally isolating said warmzone from said cold zone upon installation of the cell culture cassette,and for securing the bioreactor module solely within the warm zone andthe reagents fluid reservoir solely within the cold zone.

62. A method for maintaining a controlled thermal environment forbiological processes within a bioreactor module of a cell culturecassette, the method comprising:

creating and maintaining a distinct high velocity warm airflow at andaround the bioreactor module, and simultaneously creating andcirculating a distinct cold air flow around and through a reagents fluidreservoir operatively connected to the bioreactor module,

wherein said first airflow and said second airflow are separate andcannot intermingle.

BRIEF DESCRIPTION OF THE DRAWINGS

The following description of typical aspects described herein will bebetter understood when read in conjunction with the appended drawings.For the purpose of illustrating the invention, there are shown in thedrawings aspects which are presently typical. It should be understood,however, that the invention is not limited to the precise arrangementsand instrumentalities of the aspects shown in the drawings. It is notedthat like reference numerals refer to like elements across differentembodiments as shown in the drawings and referred to in the description.

The description herein will be more fully understood in view of thefollowing drawings:

FIG. 1 illustrates a cross section of one embodiment of a cell cultureand tissue engineering system of the invention showing a cell culturecassette engaged to operational robotics with the thermal barrierassembly in a locked position;

FIG. 2 illustrates a cross section of the system in an openconfiguration exposing the cell culture cassette that is shown with thethermal barrier assembly in an unlocked position;

FIG. 3A illustrates a front elevational view of the system showing theoperational robotics interface with the thermal barrier assembly in anupright position for installation of a cell culture cassette;

FIG. 3B illustrates a front elevational view of the system of FIG. 3Awith the thermal barrier assembly in a locked position;

FIG. 3C illustrates a close up front elevational view of the externalcold reservoir that forms part of the cold zone and is located adjacentan installed reagent fluid reservoir, the external cold reservoir hasbaffles for ensuring air return to the cold sink is not blocked. Thesebaffles align with the underlying structure of the thermal barrierassembly seen in FIG. 3B;

FIG. 4 illustrates the warm zone airflow path and features of theheating assembly;

FIG. 5 illustrates the cold zone airflow path and features of the coldthermal assembly;

FIG. 6 illustrates a close up perspective view of the reagent fluidsreservoir structure;

FIG. 7 illustrates a close up cross sectional view of the cold sinkarray;

FIG. 8 a illustrates the location of the condensation control structureadjacent the cold sink array;

FIG. 8 b illustrates an enlarged cross sectional view of thecondensation control structure;

FIG. 9 illustrates the cold thermal assembly unhinged from the system;

FIG. 10 illustrates the cold thermal assembly with unfastened cold fan;and

FIG. 11 illustrates the hot sink fan and associated cowling releasedfrom the rear of the system.

DESCRIPTION OF THE INVENTION

All publications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety. Thepublications and applications discussed herein are provided solely fortheir disclosure prior to the filing date of the present application.Nothing herein is to be construed as an admission that the presentinvention is not entitled to antedate such publication by virtue ofprior invention. In addition, the materials, methods, and examples areillustrative only and are not intended to be limiting.

In the case of conflict, the present specification, includingdefinitions, will control.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as is commonly understood by one of skill in theart to which the subject matter herein belongs. As used herein, thefollowing definitions are supplied in order to facilitate theunderstanding of the present invention.

As used herein, the articles “a” and “an” preceding an element orcomponent are intended to be non-restrictive regarding the number ofinstances (i.e. occurrences) of the element or component. Therefore, “a”or “an” should be read to include one or at least one, and the singularword form of the element or component also includes the plural unlessthe number is obviously meant to be singular.

As used herein, the terms “invention” or “present invention” arenon-limiting terms and not intended to refer to any single aspect of theparticular invention but encompass all possible aspects as described inthe specification and the claims.

As used herein the terms ‘comprises’, ‘comprising’, ‘includes’,‘including’, ‘having’ and their inflections and conjugates denote‘including but not limited to’ and are to be understood to beopen-ended, e.g., to mean including but not limited to.

As used herein, the term “about” refers to variation in the numericalquantity. In one aspect, the term “about” means within 10% of thereported numerical value. In another aspect, the term “about” meanswithin 5% of the reported numerical value. Yet, in another aspect, theterm “about” means within 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1% of thereported numerical value.

It will be understood that any component defined herein as beingincluded may be explicitly excluded from the claimed invention by way ofproviso or negative limitation.

As may be used herein the term ‘substantially’ (or synonyms thereof)denote with respect to the context a measure or extent or amount ordegree that encompass a large part or most of a referenced entity, or anextent at least moderately or much greater or larger or more effectiveor more important relative to a referenced entity or with respect to thereferenced subject matter.

As used herein the term ‘may’ denotes an option or an effect which iseither or not included and/or used and/or implemented and/or occurs, yetthe option constitutes at least a part of some embodiments of theinvention or consequence thereof, without limiting the scope of theinvention.

The phrase “and/or,” as used herein in the specification and in theclaims, should be understood to mean “either or both” of the elements soconjoined, e.g., elements that are conjunctively present in some casesand disjunctively present in other cases. Other elements may optionallybe present other than the elements specifically identified by the“and/or” clause, whether related or unrelated to those elementsspecifically identified unless clearly indicated to the contrary.

As used herein “supported”, “mounted”, “attached”, “connected”,“joined”, “coupled”, “linked” may be interchangeably used with respectto the engagement of components of the automated device of theinvention. Further, any of these terms may be used with the term“reversibly”.

As used herein, “thermal zone” should be understood to be an isolatedarea that has a defined consistent temperature in terms of being warm orin terms of being cold. Further, the zone having a defined temperatureor defined temperature range is maintained at that defined temperatureor within the defined temperature range such that that zone is stable,uniform, undeviating or homogenous with respect to the definedtemperature or defined temperature range.

As used herein, “warm zone” should be understood to be an isolatedconfined area with a precise temperature above room temperature (i.e.,above about 23° C.). Generally, the precise temperature for mammaliancells is about 37° C. However, depending on the particular needs of thespecific cell culture, temperatures above and below 37° C. may still beselected as the precise temperature and accomplished as a “warm zone”.For example, stem cells proliferate at 37° C. in the absence ofdifferentiation, if differentiation factors are absent. Conversely, stemcells growing at temperatures above and below 37° C. will differentiatewithout differentiation factors being present. Furthermore, different“warm zone” temperatures may be required for application of temperaturestress on a given culture. The “warm zone” will contain a high velocitywarm airflow path. One of skill in the art will understand the meaningof “high velocity” compared to regular airflow velocity. The “warm zone”houses the bioreactor module and has a distinct gas regulating means.

As used herein, “cold zone” should be understood to be an isolatedconfined area with a temperature range of about 2° C. to 8° C. The exacttemperature of the “cold zone” need not be precise but rather an overalltemperature reduction such as about 2° C. to 8° C. The “cold zone”contains a cold airflow. The “cold zone” houses the fluids reagentsreservoir and any further fluid reagents bags. The “cold zone” housesthe condensation control means.

The “warm zone” is separate from the “cold zone” such that thetemperature in either zone does not migrate into the other zone. Thehigh velocity warm airflow does not intermingle with the cold airflow.

The cell culture cassette resides in the warm zone.

The reagents fluids reservoir resides in the cold zone.

External (additional) fluid bags reside in the cold zone.

As used herein, “resides” with respect to the “warm zone” or the “coldzone” means that the noted structural element is only subjected to theatmosphere in that particular zone in the closed operation of theculture system.

A general non-limiting overview of the invention and practising theinvention is presented below. The overview outlines exemplary practiceof embodiments/aspects of the invention, providing a constructive basisfor variant and/or alternative and/or divergent aspects/embodiments,some of which are subsequently described.

FIGS. 1 and 2 show an embodiment of the system of the invention in aclosed and open configuration, respectively. FIG. 1 illustrates anembodiment of a cell culture and tissue engineering system 1 of theinvention in which a disposable cell culture cassette 2 is installed andoperated under automated conditions. The cell culture cassette 2comprises a bioreactor module 4 and attached reagents fluid reservoir 6.The system 1 resembles a “cocoon-shape” and comprises an inner shellbody 8 and an outer shell cover 10 that covers an opening 12 of theinner shell body. The outer shell cover 10 envelops the front opening 12of the inner shell body and is connected to the inner shell body 8 via arotatable connection (not shown) at both sides of the inner shell bodyso that the outer shell cover 10 can rotate to open along the outer arcshape of the inner shell body from a first closed position to an openposition rotating about the connection points until the outer shellcover nests the inner shell body (shown in FIG. 2 ). The front opening12 of the inner shell body 8 comprises a periphery 14 with a U shapedchannel 16 that comprises an inflatable seal 17 such that when thesystem is closed the inflatable seal is actuated to inflate and engagewith an inside surface of the outer shell body to seal the system in anairtight manner. The outer shell cover 10 comprises thermal cells 18that act as an external thermal barrier.

The cell culture cassette 2 is installed against an operational roboticsinterface 20 positioned within the opening 12 of the inner shell body 8.The operational robotics interface 20 further comprises associatedrobotics and electronics 22 within the inner shell body. The cellculture cassette 2 is operationally restrained against the operationalrobotics interface 20 and locked in this position by a movable thermalbarrier assembly 24 that extends from the cell culture cassette 2 to theouter shell cover 10. Locking the cell culture cassette 2 to theoperational robotics interface 20 by the movable thermal barrierassembly 24 creates an upper warm zone 26 and a lower cold zone 28. Thebioreactor module 4 is secured within the warm zone 26. The reagentsfluid reservoir is secured within the cold zone 28. An additionalexternal cold reservoir 29 is located within the cold zone and adjacentto the reagents fluid reservoir of an installed cell culture cassette.This external cold reservoir 29 may contain an additional reservoirbag(s) for collection of fluid waste and/or provide additional requiredculture fluid(s) and reagent(s). Thus the cell culture system providesboth a warm, incubated environment suitable for biological processes(e.g. about 37° C.+/−5° C.) and a separate cold environment for enhancedreagent stability during the period of reagent storage (e.g. over 0° C.to about 10° C.).

In FIG. 2 the system 1 is in an open configuration that exposes the cellculture cassette 2 for inspection and/or removal and further shows themovable thermal barrier assembly 24 in an open raised position. Thebioreactor module 4 of the cell culture cassette 2 is fluidly connectedto the reagent fluids reservoir 6 via mated port connections (notshown). The bioreactor module 4 supports the operational requirements ofa biological process, such as cell culture or tissue engineering andcomprises one or more bioreactors that may be operatively connected inseries. The reagent fluids reservoir 6 substantially accommodates andstores the reagents required for the biological processes occurring inthe one or more bioreactors of the bioreactor module. The reagent fluidsreservoir 6 comprises multiple flexible reagent bags (not shown), eachof the reagent bags containing one or more of culture medium, growthfactors, pharmaceutical agents, cell labels and waste productseliminated from the bioreactor module.

In operation, the outer shell cover 10 is rotated to an open position toenable access to the cassette 2 for installation or removal of the cellculture cassette that is restrained against the operational roboticsinterface 20 by way of the structural configuration of the movablebarrier assembly 24. When cell culture cassette installation/removal isrequired, the movable thermal barrier assembly 24 can be raised awayfrom the cassette by way of an internal linkage mechanism permittingfull access to the cell culture cassette. Following installation of acassette 2, the thermal barrier assembly 24 is moved to the engaged(lowered) position as shown in FIG. 1 , thereby ensuring accuratealignment with the robotics 7 and related interface connections andforming separate warm and cold zones. When the cell culture cassette isinstalled and the movable thermal barrier is locked in place, thethermal barrier effectively forms part of the lower surface of the warmzone around the cassette and the upper surface of the cold zone aroundthe cassette.

FIGS. 3A and 3B show a front elevational view of the operationalrobotics interface 20 with the movable thermal barrier assembly 24 in anopen raised position. The operational robotics interface 20 comprisesseveral valve actuators and peristaltic pump connections to which thecell culture cassette is aligned and connected with. The movable thermalbarrier assembly 24 comprises levered spaced apart arms 30 that areconnected at either side of the operational robotics interface 20 thatsupport a thermal platform 32 shown in the raised position. Theunderside of the thermal platform is shown to have channels 33. In FIG.3B the thermal platform 32 is shown with its associated upper handrail34 that closely conforms to the dimension of an installed and lockedcell culture cassette. Recesses 36 are provided adjacent the leveredarms 30 that provide operator controlled access to open and extend themovable thermal barrier assembly 24. The upper handrail 34 provides agripping means for a user to lock the cell culture cassette intoposition for operation or to unlock and raise the thermal platform 32.The thermal platform 32 is shown to extend forward and radiate aroundthe cell culture cassette to the outer shell cover (shown in FIG. 1 ).The thermal platform has both a flexible outer seal at its front outerboundary to accommodate relative motion between the thermal platform andthe outer shell cover (not shown) and a flexible inner seal that isdirectly adjacent the cell culture cassette (not shown) to accommodatemounting tolerances of the cell culture cassette. Both seals areelastomeric and impermeable to moisture and vapour.

FIG. 3C shows the inside of the external cold reservoir 38 that hasbaffles 40 to help prevent blocking of the air return to the coldthermal assembly shown at the back of the external cold reservoir. Theexternal cold reservoir 38 is positioned adjacent to the reagents fluidreservoir and both are located within the cold zone 28. Therefrigeration space may contain separate external reservoir bag(s)outside of the reagents fluid reservoir (not shown). The baffles 40 actto prevent that any reservoir bag contained in the external coldreservoir does not close off the return air back to the cold sink. Whenthe thermal platform is in the lowered locked position around the cellculture cassette the underside channels 33 line up with the baffles 40to create a continuous structure for the unobstructed cold air flow.

FIG. 4 shows the temperature-controlled warm zone 26 and cold zone 28 ofthe cell culture system 1. The system 1 is shown in the closed position.The warm zone has a substantially segregated gas environment to that ofthe cold zone. The warm zone comprises a system of gas control for gasessuch as oxygen, carbon dioxide and nitrogen. Nitrogen drives theconcentrations of oxygen and/or carbon dioxide below ambient. The warmzone 26 is created by a heating assembly 41 located in an upper sectionof the inner shell body. The heating assembly comprises a high capacitylinear fan 42 operably connected to a heated airflow director 44 togenerate and direct a high warm airflow path 45 (shown via arrows) tocirculate around the bioreactor module 4. The high capacity linear fanprovides for high velocity airflow to minimize spatial and temporaltemperature inhomogeneity within the warm zone. The linear fan 42 has aflow configuration that provides a high velocity airflow rate due to thecombination of laminar flow, minimal pressure drop and minimal airflowdirectional changes. The high airflow velocity promotes temperatureuniformity due to high convective heat transfer, which inherentlyminimizes differences in surface temperatures arising within the warmzone from competing thermal sources.

Further, continuous circulation of the high velocity warm airflow isaided by the arc shape of the inside wall of outer shell cover and helpsthe circular path of the warm high velocity airflow to be consistent andhomogeneous. The warm zone is shown to be completely thermally separatedfrom the cold zone. Competing thermal loads are placed on the warm zoneby heat transfer with other regions within the cell culture system andby heat transfer to the surrounding ambient environment. The cold zoneand typically the ambient environment tend to operate at temperaturesbelow the temperature set point of the warm zone and consequently thesefactors represent thermal losses for the warm zone. In contrast,specific electronic components within the robotic architecture mayoperate at temperatures above the temperature of the warm zone andconsequently such components represent thermal gains for the warm zone.The configuration and operation of the warm zone obviates problems ofheat transfer conditions that inherently drive temperaturenon-uniformity, such that the uniform warm zone maintains moreconsistent operating conditions of the biological processes underwaywithin the bioreactor module.

Consistent heating temperatures can be selected for the warm zone as isrequired by a particular biological process. Controlled temperatures maybe selected from 32° C., 33° C., 34° C., 35° C., 36° C., 37° C., 38° C.or more.

FIG. 5 illustrates the primary functional components of the cold zone28. The inner shell body has a floor that supports tray 46 with aforward cantilevered ledge 48 for supporting a bottom surface of thecell culture cassette forming a ducted airflow path below the tray togenerate the cold zone airflow path 49 (shown via arrows).

The cold thermal assembly 50 is contained at the rear of the inner shellbody to generate and control the cold zone airflow path surrounding thereagent fluid reservoir for enhanced reagent stability. The cold thermalassembly 50 comprises cold sink arrays 52 each comprising a plurality offin shaped cold sinks 54 (see FIG. 7 ) that receive heat from the coldzone airflow path via a cold sink fan 56. Peltier solid-statethermoelectric devices (see FIG. 7 ) transfer heat from the cold sinks54 to the adjacent hot sink 58. Heat is subsequently rejected from thehot sink to the surrounding ambient air 62 by the hot sink fan 60.

The cold sink fan 56 has an axial flow configuration that provides ahigh convective heat transfer coefficient due to turbulent flow at thehot sink fins 58 resulting in a minimal temperature difference betweenthe airflow within the refrigerated zone and the cold sink. Reagentswithin the refrigerated zone and are cooled by virtue of beingsurrounded by the cold airflow path. Temperatures within the cold zonecan be less uniform than in the warm zone, as the key criteria forenhanced reagent stability is overall temperature reduction rather thanprecise temperature accuracy.

FIG. 6 illustrates the reagent fluids reservoir 6 that stores multiplereagents in separate fluid containers such as flexible reagent bags (notshown). The reagents bags are constrained within a fluid bags container64. The reagent fluids reservoir 6 is connected to the bioreactor modulevia port connections 67 and snap tabs 66 that help to maintain theattachment to the bioreactor module. The reagent bags are insulatedagainst the migration of heat from the warm zone by the cassette thermalinsulation 68. The refrigeration temperature of the reagent bags ismaintained by the circulation of cold air surrounding and within thereagent fluid reservoir, which includes airflow above the reagent bagsvia the reagent fluids reservoir open air ducts 70 provided on the frontand back walls adjacent the top portion of the reagents fluid reservoir.The top of the reagents fluid reservoir (roof portion) has downwardlyextending baffles extending into the reservoir (not shown) that help todirect the cold airflow above and across the reagent bags and allow theairflow to move through to the external cold reservoir guided by thebaffles 40 therein to flow unobstructively back to the cold sink array.

The structure of the cold zone is such to create a ducted/channeledairflow therein with minimal obstruction to provide a continuous flow ofcold air that will circulate throughout the entire cold zone providingcold airflow underneath and surrounding the reagents fluid reservoir, aswell as having the cold air flow penetrate the top portion of thereagents fluid reservoir through the air vents for cold air to flowthrough the reservoir and directly over the fluids bags and exit throughfurther air vents into the external cold reservoir where it is directedvia baffles back to the cold sink of the cold thermal assembly to dispelheat. The provision of channels, baffles, air vents, thermal insulationand the seals and underside channels of the thermal platform togetherensure that the cold air is maintained in the cold zone with minimalobstruction for the cold air flow in order to remove heat and circulatecold air.

FIG. 7 shows the structure of the peltier solid-state thermoelectricdevices that pump heat from the cold sink to the hot sink for subsequentheat transfer to the ambient environment. Peltier solid-state devicesare preferred over other traditional methods of refrigeration such asvapour compression refrigeration because solid-state devices are compactand have no failure-prone moving parts. Thermal insulation is providedto inhibit heat from returning to the cold sink from the hot sink, assuch heat return would compromise thermal effectiveness. The cold sinkhas an adjacent incorporated monument forming an extension of the coldsink and represents a conductive path for heat to travel from the coldsink to the Peltier solid-state device. The follow-on heat deliveredfrom the Peltier solid-state device to the hot sink is the sum of theheat pumped from the cold sink and electrical power consumed by thePeltier solid-state device. As such, the aggregate heat delivered to thehot sink is significantly greater than the heat removed from the coldsink.

The monument is advantageously incorporated into the cold sink asopposed to the hot sink since less heat transfer is then requiredthrough the monument. Consequently, the temperature difference acrossthe monument is significantly less than that present if the monumentwere located on the hot sink, thereby reducing thermal losses. ForPeltier solid-state devices, the coefficient of performance (ratio ofheat pumped to electrical power consumed) increases with decreasingtemperature differential. Hence the location of the monument on the coldsink provides significant gains in the coefficient of performancerelative to the alternative of incorporating the monument as part of thehot sink.

The cold sink array 52 is comprised of multiple individual cold sinks 54relative to the hot sink 58 that is a monolith. In order to ensureintimate thermal contact between the monument of the cold sink 54, thepeltier solid-state device, and the hot sink 58, the cold sinks aresegmented into functional units (cold sink plus axial fan), whereby eachfunctional cold sink unit intimately contacts the monolith hot sink (viathe Peltier solid-state device) through the use of and array of springcompression bolts 70. The spring compression is achieved through the useof coil springs 72. The key advantage of the spring compression of thecold sink toward the monolith hot sink is that thermal distortionsand/or production distortions are self-rectified in that each cold sinkassembly can independently align and achieve homogenous compressionloads against the associated Peltier solid-state device and onwardagainst the monolith hot sink. Such self-compensating compression loadsmaximize the effectiveness of individual cold sink thermal transfer tothe monolith hot sink.

When the cell culture system is opened the cold zone inevitablyexchanges air with the ambient environment. As a result, when the cellculture system is subsequently closed, air from the ambient environmentis entrained within the cold zone. Moisture from the air of theenvironment condenses if the humidity of the incoming ambient airresults in a dew point that is above the ultimate temperature of thecold zone. The resulting inevitable condensation can generate zones ofundesirable moisture accumulation within the cold zone, as suchaccumulations can lead to a potential site for microbial contamination.Condensation naturally initiates and continues on the cold sink, as thesurface of this component is the coldest surface within therefrigeration zone. In order to automatically manage and hence removethe complications of condensation, within the cold zone the cell culturesystem employs a condensation control mechanism 73 with the locationshown in FIG. 8A. Shown more closely in FIG. 8B, the condensationcontrol mechanism 73 comprises a moisture transport material 74. Themoisture transport material 74 collects condensate as condensation formson the cold sink and travels down the fins of the cold sink by theeffect of gravity and the action of the airflow downwards over the coldsink fins. The moisture transport material protrudes from the cold sinkthrough a dedicated duct 76 to the hot sink whereupon the transmissionof moisture is subsequently and continuously evaporated into thesurrounding environment by the heat of the hot sink. This condensationmanagement strategy requires no moving parts or extra power.

Service and cleaning of the cell culture system are required for GoodManufacturing Practice. FIG. 9 shows the cold thermal assembly 50 can beunlatched and supported on a hinge 51 to hang open to enable service andcleaning. The cold thermal assembly is released from the position ofregular operation via activation of latch 78 and subsequent downwardrotation about hinge points. The refrigeration power connector 82provides for reliable electrical connections for data and power when thecold thermal assembly is returned to the normal closed operationconfiguration.

FIG. 10 further illustrates that the cold sink fan 56 comprises anassembly 84 that can be uncoupled and withdrawn away from the cold sinkto enable further detailed cleaning of the underlying cold sink.

FIG. 11 further illustrates how the hot sink fan 60 and related cowlingmay be withdrawn upwardly and away from the hot sink via hinges enablingfurther detailed cleaning of the underlying hot sink.

In embodiments, the use of a hollow shaft enables connection of theinterior of the cell culture system to the exterior of the system,allows for the creation of a unique third control zone within the shaftto enable processes to be run at a temperature other than the culturetemperature or the refrigeration temperature. Such an embodiment can beused for process steps that potentially benefit from an intermediatetemperature and can be to controlled in this transition zone.

In additional embodiments, a thermal window can be included in the warmand/or cold zones comprised of twin liquid crystal (LCD) windows (orfunctional equivalent) incorporated into the outer shell which permit:viewing of internal cassette actions when the LCD is transparent;opacity to harmful light degradation of reagent when the LCD isactivated and opaque; and a thermal barrier due to twin LCD wallsforming an entrapped air space.

In embodiments, the separate internal airflows can be linked tocentralized airflow management system capable of controlling multipleproduction units.

In additional embodiments, air filtration can be included within the aircirculation paths and such filter being disposable following eachtreatment or other reasonable period.

It is understood by one of skill in the art that where feasible,materials for fabrication of components of the system described hereinare selected to maximize thermal insulation properties withoutcompromising the primary function of the components with respect tobiological compatibility (e.g. non-toxic, USP Class VI compliant) orstructural properties (e.g. strength, rigidity, toughness and weight).While the system is shown to be generally configured in a cocoon shape,this may vary, as well as size, so long as the shape maintains the warmand cold airflow paths therein.

Furthermore the cell culture and tissue engineering system of theinvention comprises a variety of sensors associated with and/or locatedwithin the cold zone, the hot zone, the cell culture cassette, theheating assembly, the cold thermal assembly, and associated with theoperational robotics interface and associated internal robotics andelectronics and further associated with computer means.

It should be understood that various changes and modifications to thepresently preferred embodiments described herein will be apparent tothose skilled in the art. Such changes and modifications can be madewithout departing from the spirit and scope of the present subjectmatter and without diminishing its intended advantages. It is thereforeintended that such changes and modifications be covered by the appendedclaims.

1. A method for preparing a controlled thermal environment forbiological processes within an automated cell culture cassette, theautomated cell culture cassette comprising a bioreactor module and areagents fluid reservoir, the method comprising: raising a movablethermal barrier assembly into an unlocked, raised position; installingthe automated cell culture cassette into a housing having an outer shellcover and an inner shell body, and against an operational roboticsinterface positioned within the inner shell body; lowering the movablethermal barrier assembly into a locked, lowered position to restrain theautomated cell culture cassette against the operational roboticsinterface and to form a portion of a lower surface boundary of a warmzone and a portion of an upper surface boundary of a cold zone; andcirculating a warm airflow path surrounding the bioreactor module and acold airflow path surrounding the reagents fluid reservoir.
 2. Themethod of claim 1, wherein the circulating comprises circulating thewarm airflow path via a heating assembly in an upper section of theinner shell body.
 3. The method of claim 2, wherein the heating assemblyincludes a high-capacity linear fan operably connected to a heatedairflow director, and the circulating generates a consistent andhomogenous flow via the high-capacity linear fan and the heated airflowgenerator.
 4. The method of claim 2, further comprising maintaining asubstantially uniform temperature throughout the warm zone via theheating assembly.
 5. The method of claim 1, further comprisinggenerating, via a cold thermal assembly in a rear of the inner shellbody, the cold airflow path surrounding the reagents fluid reservoir. 6.The method of claim 5, wherein the cold thermal assembly generates thecold airflow path via a cold sink array that includes a plurality ofcold sinks compressed with a Peltier device to a hot sink for transferof heat to the hot sink.
 7. A cell culture system for receiving andoperationally supporting an automated cell culture cassette forbiological processes, the automated cell culture cassette comprising abioreactor module and a reagents fluid reservoir, the system comprising:a housing; an operational robotics interface; and a movable thermalbarrier assembly having an unlocked, raised position for installing theautomated cell culture cassette, and a locked, lowered position forthermally isolating a warm zone from a cold zone, the movable thermalbarrier assembly operationally restraining the automated cell culturecassette against the operational robotics interface; and wherein, whenin the locked position, the thermal barrier assembly forms a portion ofa lower surface boundary of the warm zone and a portion of an uppersurface boundary of the cold zone.
 8. The cell culture system of claim 7wherein the warm zone is configured to circulate a warm airflow pathsurrounding the bioreactor module.
 9. The cell culture system of claim8, wherein a heating assembly is provided in an upper section of thehousing, the heating assembly generating the warm airflow path directedat the bioreactor module.
 10. The cell culture system of claim 7 whereinthe cold zone is configured to circulate a cold airflow path surroundingthe reagents fluid reservoir.
 11. The cell culture system of claim 7,wherein the operational robotics interface includes valve actuators,peristaltic pump connectors, and related control systems for mating withcorresponding connections on the cell culture cassette.
 12. The cellculture system of claim 7, wherein the movable thermal barrier assemblyincludes a pair of spaced apart arms internally connected at either sideof the operational robotics interface that support a central thermalplatform with an associated upper handrail.
 13. The cell culture systemof claim 7, wherein a heating assembly generates the warm airflow pathdirected at the bioreactor module.
 14. The cell culture system of claim7, wherein a cold thermal generates and controls the cold airflow pathsurrounding the reagents fluid reservoir.
 15. The cell culture system ofclaim 7, wherein the bioreactor module is fluidly connected to thereagents fluid reservoir.
 16. The cell culture system of claim 7,wherein the cold zone further includes a condensation control.
 17. Thecell culture system of claim 7, wherein the cold zone includes a coldreservoir positioned adjacent to the reagents fluid reservoir.
 18. Thecell culture system of claim 17, wherein the cold reservoir includes atleast one baffle.
 19. The cell culture system of claim 18, wherein thethermal barrier assembly includes at least one channel, and, when thethermal barrier is in the locked position, the at least one baffle ofthe cold reservoir is aligned with the at least one channel of thethermal barrier.
 20. A cell culture system for receiving andoperationally supporting an automated cell culture cassette forbiological processes, the automated cell culture cassette comprising abioreactor module and a reagents fluid reservoir, the system comprising:a thermoelectric device to pump heat from a cold sink to a hot sink forsubsequent heat transfer to an ambient environment; thermal insulationprovided to inhibit heat from returning to the cold sink from the hotsink; an incorporated monument adjacent to the cold sink and furtherforming an extension of the cold sink, the incorporated monumentproviding a conductive path for heat to travel from the cold sink to thethermoelectric device; wherein the heat pumped from the thermoelectricdevice is a sum of the heat pumped from the cold sink, and electricpower consumed via the thermoelectric device.